Faculty Bibliography

Dopamine transporter (DAT) trafficking was assessed by functional measurements of dopamine uptake and by biotinylation of surface proteins followed by gel electrophoresis and Western blotting. In human embryonic kidney (HEK)-293 cells expressing human DAT (HEK-hDAT), pretreatment with dopamine (0.1-100 microM) followed by washout caused reductions in subsequent dopamine uptake (reflected in Vmax) with effective dopamine concentrations in the 10 to 100 microM range and pretreatment times of 10 to 60 min. Reductions assessed after 60-min pretreatment with 100 microM dopamine corresponded with decreases measured in surface DAT by the noncleavable biotin method, which were caused, at least in part, by enhanced endocytosis as monitored with cleavable biotin. Pretreatment of rat striatal synaptosomes with dopamine (10 and 100 microM) also caused reductions in DAT uptake activity (Vmax), and again the underlying mechanism seemed to be a diminished presence of DAT at the surface of synaptosomes as measured by the noncleavable biotin method. The copresence of cocaine during pretreatment with dopamine prevented the down-regulation of surface DAT. The present results show that DAT surface residency can be regulated by substrate acting on it, not only in cells heterologously expressing DAT but also in situ in rat brain tissue

Although Na+ is crucial for the function of the dopamine (DA) transporter (DAT), its role in the substrate binding step has been questioned. To address this issue, we investigated the effect of Na+ on DA binding by measuring the potency of DA in inhibiting the binding of the cocaine analogue [3H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) in intact cells expressing DAT in their plasma membranes and in membranes isolated from these cells. In cells, Na+ substantially enhanced the potency of DA in inhibiting CFT binding. This effect of Na+ was independent of buffer compositions and substitutes (sucrose vs. NMDG), more pronounced at 4 degrees C than 25 degrees C, and correlated with its stimulatory effect on DA uptake Km. Removing extracellular Na+ had little effect on intracellular concentrations of Na+ and K+, or on membrane potential. These data suggest that extracellular Na+ most likely acts at the transporter level to enhance the binding of external DA during the transport cycle. In contrast, in cell-free membrane preparations the Na+ stimulation was abolished without impairment of the potency of DA in inhibiting CFT binding, regardless of whether sucrose was used to maintain the buffer osmolarity. The difference in Na+ dependence for DA to inhibit CFT binding between plasma membranes of intact cells and isolated membranes raises the possibility that intracellular ion environment, alone or in combination with other cellular factors, plays a critical role in determining DA-DAT interaction and the integration of Na+ modulation in this interaction

To explore structure-activity relationships (SAR) of a novel conformationally constrained lead cis-3,6-disubstituted piperidine derivative derived from (2,2-diphenylethyl)-[1-(4-fluorobenzyl)piperidine-4-ylmethyl]amine (I), a series of compounds was synthesized by derivatizing the exocyclic N-atom at the 3-position of the lead. This study led to the formation of substituted phenyl and heterocyclic derivatives. All novel compounds were tested for their affinity at the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in competing for the binding of [3H]WIN 35 428, [3H]citalopram, and [3H]nisoxetine, respectively. Selected compounds were also evaluated for their activity in inhibiting the uptake of [3H]DA. The SAR results demonstrated that the nature of substitutions on the phenyl ring is important in activity at the DAT with the presence of an electron-withdrawing group having the maximum effect on potency. Replacement of the phenyl ring in the benzyl group by heterocyclic moieties resulted in the development of compounds with moderate activity for the DAT. Two most potent racemic compounds were separated by a diastereoisomeric separation procedure, and differential affinities were observed for the enantiomers. Absolute configuration of the enantiomers was obtained unambiguously by X-ray crystal structural study. One of the enantiomers, compound S,S-(-)-19a, exhibited the highest potency for the DAT (IC50 = 11.3 nM) among all the compounds tested and was as potent as GBR 12909 (1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine). However, the compound (-)-19a was more selective than GBR 12909 in binding to the DAT compared with binding to the SERT and NET. The present results establish the newly developed 3,6-disubstituted piperidine derivatives as a novel template for high-affinity inhibitors of DAT. Structurally these molecules are more constrained compared to our earlier flexible piperidine molecules and, thus, should provide more insights about their bioactive conformations

In our effort to develop high-affinity ligands for the dopamine transporter which might find potential use as cocaine medication, a polar hydroxy substituent was introduced into the piperidine ring of one of our disubstituted lead analogues derived from 1-[2-(diphenylmethoxy)-ethyl]-4-(3-phenylpropyl)piperazine (GBR 12935). Both cis- and trans-3-hydroxy derivatives were synthesized and the racemic trans isomer, (+/-)-5, was further resolved into two enantiomers. Newly synthesized compounds were characterized for their binding affinity at the dopamine, serotonin, and norepinephrine transporter systems in rat brain. The two enantiomers (+)-5 and (-)-5 exhibited marked differential affinities at the dopamine transporter with (+)-5 being 122-fold more potent than (-)-5 in inhibiting radiolabeled cocaine analogue binding (IC(50); 0.46 vs 56.7 nM) and 9-fold more active for inhibiting dopamine uptake (IC(50); 4.05 vs 38.0 nM). Furthermore, the most active (+)-5 was 22-fold more potent at the dopamine transporter compared to the standard GBR 12909. Absolute configuration of one of the enantiomers was determined unambiguously by X-ray structural analysis. In in vivo locomotor activity studies, the enantiomer (+)-5 and the racemic (+/-)-5, but not (-)-5, exhibited stimulant activity with a long duration of effect. All three compounds, (+)-5, (-)-5, and (+/-)-5, within the dose range tested, partially (50%) but incompletely (80%) produced cocaine-like responses in mice trained to discriminate 10 mg/kg ip cocaine from vehicle. Compound (-)-5 was distinctive in this regard in that, unlike (+)-5 and (+/-)-5, it did not affect locomotor activity yet, but similar to them, was able to engender (albeit incompletely) cocaine-like responses

1. Conflicting results have been reported regarding the influence of nitric oxide (NO) and peroxynitrite on dopamine (DA) uptake and release. In the present study, effects of NO donors were studied in rat C6 glioma cells expressing human DA transporter. 2. [(3)H]-DA uptake was inhibited by S-nitroso-thiol S-nitroso-N-acetylpenicillamine, spermine/NO, diethylamine/NO (DEA/NO), (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)-amino]/NO (PAPA/NO), and 3-morphosynodiomine (SIN-1) in a rank order correlating with their half lives as NO donors, whereas no effect was observed for diethylenetriamine/NO and dipropylenetriamine/NO, which release NO very slowly. 3. Hydroxycobalamin, a NO scavenger, but not superoxide dismutase and catalase, enzymes that metabolize superoxide and hydrogen peroxide, respectively, abolished the inhibitory effect of DEA/NO and SIN-1, indicating that they inhibit DA uptake through a mechanism related to the production of NO but unrelated to the formation of peroxynitrite. In consonance, peroxynitrite did not alter DA uptake in the present system. 4. DEA/NO and PAPA/NO reduced [(3)H]-MPP(+) uptake, whereas the release of [(3)H]-MPP(+) was not modified, demonstrating that NO can inhibit uptake of DA transporter substrate without accelerating DA transporter-mediated reverse transport of substrate under the same conditions

2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) has been increasingly used as nitric oxide (NO) scavenger. Carboxy-PTIO reacts with NO to form nitric dioxide and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl (carboxy-PTI). In rat C6 glioma cells expressing human dopamine transporter, carboxy-PTIO paradoxically potentiated the inhibition of [(3)H]dopamine uptake by two NO donors, diethylamine/NO and (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)-amino]/NO. Further examinations revealed that carboxy-PTI concentration-dependently reduced dopamine uptake, indicating that the formation of carboxy-PTI may account for the failure of carboxy-PTIO to abolish NO elicited effects. These results suggest that caution should be taken in interpreting data obtained using carboxy-PTIO and probably other NO scavengers

Receptor-mediated feedback control plays an important role in dopamine (DA) neurotransmission. Recent evidence suggests that release and uptake, key mechanisms determining brain extracellular levels of the neurotransmitter, are governed by presynaptic autoreceptors. The goal of this study was to investigate whether autoreceptors regulate both mechanisms concurrently. Extracellular DA in the caudate-putamen and nucleus accumbens, evoked by electrical stimulation of the medial forebrain bundle, was monitored in the anesthetized rat by real-time voltammetry. Effects of the D2 antagonist haloperidol (0.5 mg/kg, i.p.) on evoked DA levels were measured to evaluate autoreceptor control mechanisms. Two strategies were used to resolve individual contributions of release and uptake to the robust increases in DA signals observed after acute haloperidol challenge in naive animals: pretreatment with 3beta-(p-chlorophenyl)tropan-2beta-carboxylic acid p-isothiocyanatophenylmethyl ester hydrochloride (RTI-76; 100 nmol, i.c.v.), an irreversible inhibitor of the DA transporter, and kinetic analysis of extracellular DA dynamics. RTI-76 effectively removed the uptake component from recorded signals. In RTI-76-pretreated rats, haloperidol induced only modest increases in DA elicited by low frequencies and had little or no effect at high frequencies. These results suggest that D2 antagonism alters uptake at all frequencies but only release at low frequencies. Kinetic analysis similarly demonstrated that haloperidol decreased V(max) for DA uptake and increased DA release at low (10-30 Hz) but not high (40-60 Hz) stimulus frequencies. We conclude that presynaptic DA autoreceptors concurrently downregulate release and upregulate uptake, and that the mechanisms are also independently controlled during neurotransmission

In membrane preparations, CFT, a phenyltropane cocaine analog, and dopamine (DA) interact with the recombinant human dopamine transporter (hDAT) in Na+ -free medium. Na+ markedly increased the transporter's affinity for CFT, but had little or no effect on DA potency for inhibiting CFT binding. Raising [Na+ ] from 20 to 155 mm reduced Li+ -induced increase in DA K (i), but not CFT K (d). The presence of 155 mm Na+ enhanced the tolerance to low pH of CFT Kd but not DA Ki. Leucine substitution for tryptophan 84 (W84L) in transmembrane domain (TM) 1 or asparagine substitution for aspartate 313 (D313N) in TM 6 did not or only modestly enhance the affinity of Na+ -independent CFT binding, and retained the near normal ability of DA, Li+, K+, or H+ to inhibit this binding. However, the mutations significantly enhanced the Na+ stimulation of CFT binding as well as the Na+ antagonism against Li+ and H+ inhibition of CFT binding. In contrast, the mutations neither changed the Na+ -insensitive feature of DA Ki nor enhanced the Na+ protection of DA Ki against Li+ 's inhibitory effect, though they caused Na+ protection of DA Ki against H+ 's inhibitory action. These results are consistent with the existence of binding conformations for DA that are distinguishable from those for CFT, and with a differential association of cation interactions with DA and CFT binding. The mutations likely alter Na+ -bound state(s) of hDAT, preferentially strengthening the positive allosteric coupling between Na+ and CFT binding, and reducing the impact of Li+ or H+ on the CFT binding

The role of Na(+) in the recognition of blockers by the dopamine transporter is accomodated by a model with a cation site that overlaps with the blocker binding domain, and a distal Na(+) site that interacts with this cation site and perhaps with the blocker binding domain itself. The present study addresses the application of this model to the recognition of substrates by the dopamine transporter, focusing on conditions that should reveal a stimulatory effect, if present, of Na(+) on substrate binding. Recognition was studied via the inhibition of binding of [(3)H]WIN 35,428 (2beta-carbomethoxy-3beta-(4-fluorophenyl) [(3)H]tropane), a cocaine analog, to the human dopamine transporter in human embryonic kidney 293 cells. Little or no changes in binding were noted for dopamine, d-amphetamine, p-tyramine, or dl-octopamine by increasing [Na(+)] from 2 mM to 20 mM with co-varying Br(-), both at pH 7.4 and 7.0. In 74-mM Tris-HBr or -HCl, only dopamine and d-amphetamine showed binding increases upon raising Na(+), leveling off with NO(3)(-) or SO(4)(2-) but not Br(-) as anion at approximately 60 mM Na(+), consonant with a partly stimulatory action of Br(-). An Na(+) free, low 5-mM Tris-HEPES buffer was used for studying Na(+) curves truly starting at 0 mM, and, with SO(4)(2-) as the anion, no stimulation of binding by Na(+) was observed. This suggested that the stimulations observed in high (74 mM) Tris(+) buffer by Na(+) were not a direct effect of Na(+) but rather a disinhibitory effect of Na(+) in removing Tris(+) inhibition that depended upon substrate. Tris(+) IC(50) values in Na(+) free buffer were not lower for dopamine and d-amphetamine than p-tyramine and dl-octopamine. No evidence was found for a stronger inhibitory effect of Na(+) for dopamine and dl-octopamine potentially offsetting Tris(+) disinhibition. All results together support the existence of a substrate domain overlapping with a cation site that also binds Tris(+); a distal Na(+) site interacts with this cation site and with the substrate domain by negative allosterism and is additionally impacted by Cl(-). Importantly, interactions between sites vary with the type of substrate, and, in membrane preparations, Na(+) is not required for, or stimulatory to, the binding of any of the four substrates studied unlike the binding of the cocaine analog WIN 35,428